Evolution of pineal gland: "The defining feature of the pineal gland is the capacity to function as a melatonin factory that operates on a approximately 24 h schedule, reflecting the unique synthetic capacities of the pinealocyte. Melatonin synthesis is typically elevated at night and serves to provide the organism with a signal of nighttime. Melatonin levels can be viewed as hands of the clock. Issues relating to the evolutionary events leading up to the immergence of this system have not received significant attention. When did melatonin synthesis appear in the evolutionary line leading to vertebrates? When did a distinct pineal gland first appear? What were the forces driving this evolutionary trend? As more knowledge has grown about the pinealocyte and the relationship it has to retinal photoreceptors, it has become possible to generate a plausible hypothesis to explain how the pineal gland and the melatonin rhythm evolved. At the heart of the hypothesis is the melatonin rhythm enzyme arylalkylamine N-acetyltransferase (AANAT). The advances supporting the hypothesis will be reviewed here and expanded beyond the original foundation;the hypothesis and its implications will be addressed." From (1) AANAT E-Box: "Arylalkylamine N-acetyltransferase (Aanat) is the penultimate enzyme in the serotonin-N-acetylserotonin-melatonin pathway. It is nearly exclusively expressed in the pineal gland and the retina. A marked rhythm of Aanat gene expression in the rat pineal is mediated by cyclic AMP response elements located in the promoter and first intron. Intron 1 also contains E-box elements, which mediate circadian gene expression in other cells. Here we examined whether these elements contribute to rhythmic Aanat expression in the pineal gland. This was done using transgenic rats carrying Aanat transgenes with mutant E-box elements. Circadian expression of Aanat transgenes was not altered by these mutations. However, these mutations enhanced ectopic expression establishing that the intronic Aanat E-box elements contribute to the gene's pineal specific expression. A similar role of the Aanat E-box has been reported in zebrafish, indicating that Aanat E-box mediated silencing is a conserved feature of vertebrate biology." From (2). "In all ..... species, AANAT activity is regulated at the post-translational level and, to a variable degree, also at the transcriptional level. Here, the transcriptional regulation of pineal aanat (aanat2) of the gilthead seabream (Sparus aurata) was investigated. Real-time polymerase chain reaction quantification of aanat2 mRNA levels in the pineal gland collected throughout the 24-h cycle revealed a rhythmic expression pattern. In cultured pineal glands, the amplitude was reduced, but the daily rhythmic expression pattern was maintained under constant illumination, indicating a circadian clock-controlled regulation of seabream aanat2 In NIH-3T3 cells, the seabream aanat2 promoter was activated by a synergistic action of BMAL/CLOCK and orthodenticle homeobox 5 (OTX5). Promoter sequence analyses revealed the presence of the photoreceptor conserved element and an extended E-box (i.e. the binding sites for BMAL/CLOCK and OTX5 that have been previously associated with pineal-specific and rhythmic gene expression). These results suggest that seabream aanat2 is a clock-controlled gene that is regulated by conserved mechanisms." (From 3) NeuroD: "NeuroD1/BETA2, a member of the bHLH transcription factor family, is known to influence the fate of specific neuronal, endocrine and retinal cells. We report here that NeuroD1 mRNA is highly abundant in the developing and adult rat pineal gland. Pineal expression begins in the 17-day embryo at which time it is also detectable in other brain regions. Expression in the pineal gland increases during the embryonic period and is maintained thereafter at levels equivalent to those found in the cerebellum and retina. In contrast, NeuroD1 mRNA decreases markedly in non-cerebellar brain regions during development. Pineal NeuroD1 levels are similar during the day and night, and do not appear to be influenced by sympathetic neural input. Gene expression analysis of the pineal glands from neonatal NeuroD1 knockout mice identifies 127 transcripts that are down-regulated (>twofold, p <0.05) and 16 that are up-regulated (>twofold, p <0.05). According to quantitative RT-PCR, the most dramatically down-regulated gene is kinesin family member 5C ( approximately 100-fold) and the most dramatically up-regulated gene is glutamic acid decarboxylase 1 ( approximately fourfold). Other impacted transcripts encode proteins involved in differentiation, development, signal transduction and trafficking. These findings represent the first step toward elucidating the role of NeuroD1 in the rodent pinealocyte." From (4). Crx/Otx: "Otx2 is a vertebrate homeobox gene, which has been found to be essential for the development of rostral brain regions and appears to play a role in the development of retinal photoreceptor cells and pinealocytes. In this study, the temporal expression pattern of Otx2 was revealed in the rat brain, with special emphasis on the pineal gland throughout late embryonic and postnatal stages. Widespread high expression of Otx2 in the embryonic brain becomes progressively restricted in the adult to the pineal gland. Crx (cone-rod homeobox), a downstream target gene of Otx2, showed a pineal expression pattern similar to that of Otx2, although there was a distinct lag in time of onset. Otx2 protein was identified in pineal extracts and found to be localized in pinealocytes. Total pineal Otx2 mRNA did not show day-night variation, nor was it influenced by removal of the sympathetic input, indicating that the level of Otx2 mRNA appears to be independent of the photoneural input to the gland. Our results are consistent with the view that pineal expression of Otx2 is required for development and we hypothesize that it plays a role in the adult in controlling the expression of the cluster of genes associated with phototransduction and melatonin synthesis." From (5) Pax4: "Pax 4 is a homeobox gene encoding a transcription factor that is essential for embryonic development of the endocrine pancreas. In the pancreas, Pax4 counters the effects of the related Pax6 protein, which is also involved in development of the retina and the pineal gland. In this study, we report that Pax4 is strongly expressed in the pineal gland and retinal photoreceptors of the rat. Pineal and retinal Pax4 mRNA is low in the fetus and increases postnatally;Pax6 exhibits an inverse pattern of expression, being very strongly expressed in both tissues in the fetus. In the adult, the abundance of Pax4 mRNA exhibits a diurnal rhythm in both the pineal gland and retina with maximal levels occurring late in the day. Sympathetic denervation of the pineal gland by superior cervical ganglionectomy prevents the nocturnal decrease in pineal Pax4 mRNA but does not alter the pattern of retinal Pax4 expression. Norepinephrine is released by sympathetic nerves in the pineal gland at night;in the present study, we found that treatment with adrenergic agonists suppresses pineal Pax4 expression in vivo and in vitro. Norepinephrine is known to elevate pineal cyclic AMP;here it was found that treatment with a cyclic AMP mimic reduces pineal Pax4 mRNA. These findings suggest that the nocturnal decrease in pineal Pax4 mRNA is controlled by an adrenergic-cyclic AMP mechanism and that Pax4 may function to mediate adrenergic control of circadian gene expression in the pineal gland." (Rath et al., in preparation). Eya2. Eya2, The homolog of the Drosophila gene eyeless has been identified in the pineal gland and retina. Efforts are underway to characterize the gene products, expression pattern and function.